Sample preparation for difficult sample types

ABSTRACT

Devices and methods are provided for collecting and handling difficult sample types.

RELATED APPLICATIONS DATA

This application is a divisional application of U.S. patent applicationSer. No. 15/340,612, filed Nov. 1, 2016, which claims the benefit, under35 U.S.C. § 119(e), of U.S. Provisional Application Ser. No. 62/249,592,filed Nov. 2, 2015, the entire contents of each of which areincorporated by reference herein.

BACKGROUND 1. Technical Field

Embodiments of the present disclosure relate generally to methods anddevices for extracting nucleic acids from a sample.

2. Background

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms or viruses and the low levels of organism or viruspresent in some pathological specimens. It is often impractical to runlarge panels of PCR assays, one for each possible causative organism orviruses, most of which are expected to be negative. The problem isexacerbated when pathogen nucleic acid is at low concentration andrequires a large volume of sample to gather adequate reaction templates.In some cases there is inadequate sample to assay for all possibleetiological agents. A solution is to run “multiplex PCR” wherein thesample is concurrently assayed for multiple targets in a singlereaction. While multiplex PCR has proved to be valuable in some systems,shortcomings exist concerning robustness of high level multiplexreactions and difficulties for clear analysis of multiple products. Tosolve these problems, the assay may be subsequently divided intomultiple secondary PCRs. Nesting secondary reactions within the primaryproduct increases robustness. Closed systems such as the FilmArray®(BioFire Diagnostics, LLC, Salt Lake City, Utah) reduce handling,thereby diminishing contamination risk.

Sample preparation is often a balance between harsh extraction andlysing conditions for releasing nucleic acids from tougher materialssuch as spores and paraffin preserved samples, and gentler lysingconditions that may minimize nucleic acid degradation, particularly incontaminants that lyse more easily and have longer chromosomes. It wouldbe desirable to be able to extract nucleic acids from tougher materialswithout degrading other nucleic acids that may be present in the sample.

In addition, certain sample types can be difficult to handle. It can bedifficult to introduce a solid, semi-solid, or viscous sample to abiological processing system. Sample types such as sputum are difficultto pipette and measure, and often require pretreatment for a period oftime prior to sample processing, illustratively with heat ordithiothreitol, to reduce viscosity and help break up the sample matrix.In addition to sputum, other difficult biological samples include butare not limited to mucus, BAL, and other respiratory sample types,stool, tissue, tissue homogenate, ground tissue, paraffin treatedformalin embedded tissue, bone, bone homogenate, eschars, puss, synovialfluid, lymph node aspirates, and stomach washings. Environmentalsamples, illustratively soil, surfaces, powders or food, are often solidor semi-solid and may also present challenges. Moreover, extensivehandling during sample pre-treatment can lead to cross-contamination,can be time consuming, and it often dilutes the sample, often leading todecreased sensitivity.

The present invention addresses various issues relating to preparationof a sample prior to further testing, for example for purification ofnucleic acids from a sample, for biological analysis.

SUMMARY OF THE INVENTION

In one aspect of the present disclosure, a sample collection method isprovided, the method comprising collecting a sample comprising abiological or environmental substance using a swab, placing the swabwith the collected sample in a sample buffer, and filtering the sample.In various illustrative embodiments, the sample buffer may include aprotease or a detergent and the sample may be collected and transferredto the sample buffer using a flocked swab.

In another aspect, a sample collection method is provided, the methodcomprising collecting a sample comprising a biological or environmentalsubstance, and placing the sample in a sample buffer wherein the samplebuffer comprises a detergent in an amount of at least 10% by volume ofthe sample buffer. Illustrative embodiments may include drawing thesample through a filter. In one more aspect, a sample collection methodis provided, the method comprising collecting a sample comprising abiological or environmental substance by contacting the sample with amaterial that preferentially collects and releases the organism oversample matrix, and placing the material with the collected sample in asample buffer. Illustratively, the material is a hydrophilic material oradsorbent material. In one embodiment, a fixed amount of the materialreproducibly absorbs and releases an amount of the sample within afour-fold range.

In another aspect of the disclosure a cannulated vial is provided, thecannulated vial comprising a vial body having a top surface at one end,a bottom surface at an opposite end, and exterior wall therebetweendefining an interior vial volume, the top surface having an opening, acannula extending from the bottom surface and having a first end, asecond end and an outer surface therebetween defining a cannula volume,the first end in fluid communication with the interior vial volume, anda filter disposed between the vial body and the cannula, and an additiveprovided in the cannulated vial.

In yet another aspect of the disclosure, a sample container is provided,the sample container comprising an opening configured to receive asample, a body configured to hold a fluid, an opening configured toallow the fluid to exit, a filter disposed between the body and theexit, and an additive provided for treating the sample. Illustratively,the additive may be provided dried in the sample container. Illustrativeadditives include proteases, DNAses, DNAse inhibitors, RNAses, RNAseinhibitors, and lysozymes.

In still another aspect of the disclosure, a method of introducing anadditive to a system is provided, the method comprising obtaining thesample container as described above, the sample container holding thefluid, introducing the sample through the opening into the fluid,drawing the fluid through the filter and out the exit.

In one more aspect, methods of amplifying nucleic acids from a directblood sample are provided, the methods comprising adding the directblood sample to a sample buffer, bead beating the sample buffer,extracting the nucleic acids from the sample buffer, and amplifying thenucleic acids. Illustratively, such methods may be performed in a closedsample vessel. In another illustrative method, the steps are performedwithout one or more of centrifugation, ethanol precipitation, and DNAdigestion.

Additional features and advantages of the embodiments of the inventionwill be set forth in the description which follows or may be learned bythe practice of such embodiments. The features and advantages of suchembodiments may be realized and obtained by means of the instruments andcombinations particularly pointed out in the appended claims. These andother features will become more fully apparent from the followingdescription and appended claims, or may be learned by the practice ofsuch embodiments as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the invention can be obtained, a moreparticular description of the invention briefly described above will berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 shows a flexible pouch according to one embodiment of the presentinvention.

FIG. 2 is an exploded perspective view of an instrument for use with thepouch of FIG. 1, including the pouch of FIG. 1, according to an exampleembodiment of the present invention.

FIG. 3 shows a partial cross-sectional view of the instrument of FIG. 2,including the bladder components of FIG. 2, with the pouch of FIG. 1shown in dashed lines, according to an example embodiment of the presentinvention.

FIG. 4 shows a motor used in one illustrative embodiment of theinstrument of FIG. 2.

FIG. 5 shows a loading station for loading the pouch of FIG. 1,including the pouch of FIG. 1, according to an example embodiment of thepresent invention.

FIG. 6 shows a sample vial for loading a sample into the pouch of FIG.1.

FIG. 7 shows a hydration vial for providing a hydration fluid to thepouch of FIG. 1, according to an example embodiment of the presentinvention.

FIG. 8 shows a loading station comparable to FIG. 5, but displaying adifferent loading station configuration and vials for use with theloading station, according to an example embodiment of the presentinvention.

FIG. 9 shows a portion of the sample vial of FIG. 8 and how the samplevial keys to the sample vial receptacle of loading station of FIG. 8,according to an example embodiment of the present invention.

FIG. 10 shows a portion of a hydration vial of FIG. 8 and how thehydration vial keys to the hydration vial receptacle of loading stationof FIG. 8, according to an example embodiment of the present invention.

DETAILED DESCRIPTION

Example embodiments are described below with reference to theaccompanying drawings. Many different forms and embodiments are possiblewithout deviating from the spirit and teachings of this disclosure andso the disclosure should not be construed as limited to the exampleembodiments set forth herein. Rather, these example embodiments areprovided so that this disclosure will be thorough and complete, and willconvey the scope of the disclosure to those skilled in the art. In thedrawings, the sizes and relative sizes of layers and regions may beexaggerated for clarity. Like reference numbers refer to like elementsthroughout the description.

Unless defined otherwise, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which the present disclosure pertains.It will be further understood that terms, such as those defined incommonly used dictionaries, should be interpreted as having a meaningthat is consistent with their meaning in the context of the presentapplication and relevant art and should not be interpreted in anidealized or overly formal sense unless expressly so defined herein. Theterminology used in the description of the invention herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. While a number of methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present disclosure, only certain exemplary materials andmethods are described herein.

All publications, patent applications, patents or other referencesmentioned herein are incorporated by reference for in their entirety. Incase of a conflict in terminology, the present specification iscontrolling.

Various aspects of the present disclosure, including devices, systems,methods, etc., may be illustrated with reference to one or moreexemplary implementations. As used herein, the terms “exemplary” and“illustrative” mean “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other implementations disclosed herein. In addition, reference toan “implementation” or “embodiment” of the present disclosure orinvention includes a specific reference to one or more embodimentsthereof, and vice versa, and is intended to provide illustrativeexamples without limiting the scope of the invention, which is indicatedby the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a tile” includes one, two, or more tiles. Similarly,reference to a plurality of referents should be interpreted ascomprising a single referent and/or a plurality of referents unless thecontent and/or context clearly dictate otherwise. Thus, reference to“tiles” does not necessarily require a plurality of such tiles. Instead,it will be appreciated that independent of conjugation; one or moretiles are contemplated herein.

As used throughout this application the words “can” and “may” are usedin a permissive sense (i.e., meaning having the potential to), ratherthan the mandatory sense (i.e., meaning must). Additionally, the terms“including,” “having,” “involving,” “containing,” “characterized by,”variants thereof (e.g., “includes,” “has,” “involves,” “contains,”etc.), and similar terms as used herein, including the claims, shall beinclusive and/or open-ended, shall have the same meaning as the word“comprising” and variants thereof (e.g., “comprise” and “comprises”),and do not exclude additional, un-recited elements or method steps,illustratively.

As used herein, directional and/or arbitrary terms, such as “top,”“bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,”“outer,” “internal,” “external,” “interior,” “exterior,” “proximal,”“distal,” “forward,” “reverse,” and the like can be used solely toindicate relative directions and/or orientations and may not beotherwise intended to limit the scope of the disclosure, including thespecification, invention, and/or claims.

It will be understood that when an element is referred to as being“coupled,” “connected,” or “responsive” to, or “on,” another element, itcan be directly coupled, connected, or responsive to, or on, the otherelement, or intervening elements may also be present. In contrast, whenan element is referred to as being “directly coupled,” “directlyconnected,” or “directly responsive” to, or “directly on,” anotherelement, there are no intervening elements present.

Example embodiments of the present inventive concepts are describedherein with reference to cross-sectional illustrations that areschematic illustrations of idealized embodiments (and intermediatestructures) of example embodiments. As such, variations from the shapesof the illustrations as a result, for example, of manufacturingtechniques and/or tolerances, are to be expected. Thus, exampleembodiments of the present inventive concepts should not be construed aslimited to the particular shapes of regions illustrated herein but areto include deviations in shapes that result, for example, frommanufacturing. Accordingly, the regions illustrated in the figures areschematic in nature and their shapes are not intended to illustrate theactual shape of a region of a device and are not intended to limit thescope of example embodiments.

It will be understood that although the terms “first,” “second,” etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element could be termed a“second” element without departing from the teachings of the presentembodiments.

It is also understood that various implementations described herein canbe utilized in combination with any other implementation described ordisclosed, without departing from the scope of the present disclosure.Therefore, products, members, elements, devices, apparatus, systems,methods, processes, compositions, and/or kits according to certainimplementations of the present disclosure can include, incorporate, orotherwise comprise properties, features, components, members, elements,steps, and/or the like described in other implementations (includingsystems, methods, apparatus, and/or the like) disclosed herein withoutdeparting from the scope of the present disclosure. Thus, reference to aspecific feature in relation to one implementation should not beconstrued as being limited to applications only within saidimplementation.

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims. To facilitate understanding, like reference numerals have beenused, where possible, to designate like elements common to the figures.Furthermore, where possible, like numbering of elements have been usedin various figures. Furthermore, alternative configurations of aparticular element may each include separate letters appended to theelement number.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 5%. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; a solution containing one or moremolecules derived from a cell, cellular material, or viral material(e.g. a polypeptide or nucleic acid); or a solution containing anon-naturally occurring nucleic acid, which is assayed as describedherein. A sample may also be any body fluid or excretion (for example,but not limited to, blood, urine, stool, saliva, tears, bile, orcerebrospinal fluid) that may or may not contain host or pathogen cells,cell components, or nucleic acids.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof.

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandednucleic acid molecule of defined sequence that can base-pair to a secondnucleic acid molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, and the extent of the base-pairing that occurs. Theextent of base-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes, primers, and oligonucleotides may be detectably-labeled, eitherradioactively, fluorescently, or non-radioactively, by methodswell-known to those skilled in the art. dsDNA binding dyes may be usedto detect dsDNA. It is understood that a “primer” is specificallyconfigured to be extended by a polymerase, whereas a “probe” or“oligonucleotide” may or may not be so configured.

By “dsDNA binding dyes” is meant dyes that fluoresce differentially whenbound to double-stranded DNA than when bound to single-stranded DNA orfree in solution, usually by fluorescing more strongly. While referenceis made to dsDNA binding dyes, it is understood that any suitable dyemay be used herein, with some non-limiting illustrative dyes describedin U.S. Pat. No. 7,387,887, herein incorporated by reference. Othersignal producing substances may be used for detecting nucleic acidamplification and melting, illustratively enzymes, antibodies, etc., asare known in the art.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant typically occur at aboutmelting temperature (Tm) minus 5° C. (i.e. 5° below the Tm of theprobe). Functionally, high stringency conditions are used to identifynucleic acid sequences having at least 80% sequence identity.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependent amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. For amplification methodswithout discrete cycles, reaction time may be used where measurementsare made in cycles or Cp, and additional reaction time may be addedwhere additional PCR cycles are added in the embodiments describedherein. It is understood that protocols may need to be adjustedaccordingly.

While various examples herein reference human targets and humanpathogens, these examples are illustrative only. Methods, kits, anddevices described herein may be used to detect and sequence a widevariety of nucleic acid sequences from a wide variety of samples,including, human, veterinary, industrial, and environmental.

Various embodiments disclosed herein use a self-contained nucleic acidanalysis pouch to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. Such systems, includingpouches and instruments for use with the pouches, are disclosed in moredetail in U.S. Pat. Nos. 8,394,608; and 8,895,295; and U.S. PatentApplication No. 2014-0283945, herein incorporated by reference. However,it is understood that such pouches are illustrative only, and thenucleic acid preparation and amplification reactions discussed hereinmay be performed in any of a variety of open or closed system samplevessels as are known in the art, including 96-well plates, plates ofother configurations, arrays, carousels, and the like, using a varietyof nucleic acid purification and amplification systems, as are known inthe art. While the terms “sample well”, “amplification well”,“amplification container”, or the like are used herein, these terms aremeant to encompass wells, tubes, and various other reaction containers,as are used in these amplification systems. In one embodiment, the pouchis used to assay for multiple pathogens. The pouch may include one ormore blisters used as sample wells, illustratively in a closed system.Illustratively, various steps may be performed in the optionallydisposable pouch, including nucleic acid preparation, primary largevolume multiplex PCR, dilution of primary amplification product, andsecondary PCR, culminating with optional real-time detection orpost-amplification analysis such as melting-curve analysis. Further, itis understood that while the various steps may be performed in pouchesof the present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

FIG. 1 shows an illustrative pouch 510 that may be used in variousembodiments, or may be reconfigured for various embodiments. Pouch 510is similar to FIG. 15 of U.S. Pat. No. 8,895,295, with like itemsnumbered the same. Fitment 590 is provided with entry channels 515 athrough 515 l, which also serve as reagent reservoirs or wastereservoirs. Illustratively, reagents may be freeze dried in fitment 590and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566,with their respective channels 514, 538, 543, 552, 553, 562, and 565 aresimilar to blisters of the same number of FIG. 15 of U.S. Pat. No.8,895,295. Second-stage reaction zone 580 of FIG. 1 is similar to thatof U.S. Pat. No. 8,895,295, but the second-stage wells 582 of highdensity array 581 are arranged in a somewhat different pattern. The morecircular pattern of high density array 581 of FIG. 1 eliminates wells incorners and may result in more uniform filling of second-stage wells582. As shown, the high density array 581 is provided with 102second-stage wells 582. Pouch 510 is suitable for use in the FilmArray®instrument (BioFire Diagnostics, LLC, Salt Lake City, Utah). However, itis understood that the pouch embodiment is illustrative only.

While other containers may be used, illustratively, pouch 510 is formedof two layers of a flexible plastic film or other flexible material suchas polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene, polymethylmethacrylate, and mixtures thereof that can bemade by any process known in the art, including extrusion, plasmadeposition, and lamination. Metal foils or plastics with aluminumlamination also may be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing. Illustratively, the material has low nucleic acidbinding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by testingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in second-stage wells 582 of thesecond-stage reaction zone 580 of pouch 510, then one or both layers atwells 582 would be left without the foil. In the example of PCR, filmlaminates composed of polyester (Mylar, Dupont, Wilmington Del.) ofabout 0.0048 inch (0.1219 mm) thick and polypropylene films of0.001-0.003 inch (0.025-0.076 mm) thick perform well. Illustratively,pouch 510 is made of a clear material capable of transmittingapproximately 80%-90% of incident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpressure, the pouch material illustratively is flexible enough to allowthe pressure to have the desired effect. The term “flexible” is hereinused to describe a physical characteristic of the material of pouch. Theterm “flexible” is herein defined as readily deformable by the levels ofpressure used herein without cracking, breaking, crazing, or the like.For example, thin plastic sheets, such as SaranTM wrap and Ziploc® bags,as well as thin metal foil, such as aluminum foil, are flexible.However, only certain regions of the blisters and channels need beflexible, even in embodiments employing pneumatic pressure. Further,only one side of the blisters and channels need to be flexible, as longas the blisters and channels are readily deformable. Other regions ofthe pouch 510 may be made of a rigid material or may be reinforced witha rigid material.

Illustratively, a plastic film is used for pouch 510. A sheet of metal,illustratively aluminum, or other suitable material, may be milled orotherwise cut, to create a die having a pattern of raised surfaces. Whenfitted into a pneumatic press (illustratively A-5302-PDS, JanesvilleTool Inc., Milton Wis.), illustratively regulated at an operatingtemperature of 195° C., the pneumatic press works like a printing press,melting the sealing surfaces of plastic film only where the die contactsthe film. Various components, such as PCR primers (illustrativelyspotted onto the film and dried), antigen binding substrates, magneticbeads, and zirconium silicate beads may be sealed inside variousblisters as the pouch 510 is formed. Reagents for sample processing canbe spotted onto the film prior to sealing, either collectively orseparately. In one embodiment, nucleotide tri-phosphates (NTPs) arespotted onto the film separately from polymerase and primers,essentially eliminating activity of the polymerase until the reaction ishydrated by an aqueous sample. If the aqueous sample has been heatedprior to hydration, this creates the conditions for a true hot-start PCRand reduces or eliminates the need for expensive chemical hot-startcomponents.

Pouch 510 may be used in a manner similar to that described in U.S. Pat.No. 8,895,295. In one illustrative embodiment, a 300 μl mixturecomprising the sample to be tested (100 μl) and lysis buffer (200 μl) isinjected into an injection port (not shown) in fitment 590 near entrychannel 515 a, and the sample mixture is drawn into entry channel 515 a.Water is also injected into a second injection port (not shown) of thefitment 590 adjacent entry channel 515 l, and is distributed via achannel (not shown) provided in fitment 590, thereby hydrating up toeleven different reagents, each of which were previously provided in dryform at entry channels 515 b through 515 l via. These reagentsillustratively may include freeze-dried PCR reagents, DNA extractionreagents, wash solutions, immunoassay reagents, or other chemicalentities. Illustratively, the reagents are for nucleic acid extraction,first-stage multiplex PCR, dilution of the multiplex reaction, andpreparation of second-stage PCR reagents, as well as control reactions.In the embodiment shown in FIG. 1, all that need be injected is thesample solution in one injection port and water in the other injectionport. After injection, the two injection ports may be sealed. For moreinformation on various configurations of pouch 510 and fitment 590, seeU.S. Pat. No. 8,895,295, already incorporated by reference.

After injection, the sample is moved from injection channel 515 a tolysis blister 522 via channel 514. Lysis blister 522 is provided withbeads or particles 534, such as ceramic beads, and is configured forvortexing via impaction using rotating blades or paddles provided withinthe FilmArray® instrument. Bead-milling, by shaking or vortexing thesample in the presence of lysing particles such as zirconium silicate(ZS) beads 534, is an effective method to form a lysate. It isunderstood that, as used herein, terms such as “lyse,” “lysing,” and“lysate” are not limited to rupturing cells, but that such terms includedisruption of non-cellular particles, such as viruses.

FIG. 4 shows a bead beating motor 819, comprising blades 821 that may bemounted on a first side 811 of support member 802, of instrument 800shown in FIG. 2. Blades may extend through slot 804 to contact pouch510. It is understood, however, that motor 819 may be mounted on otherstructures of instrument 800. In one illustrative embodiment, motor 819is a Mabuchi RC-280SA-2865 DC Motor (Chiba, Japan), mounted on supportmember 802. In one illustrative embodiment, the motor is turned at 5,000to 25,000 rpm, more illustratively 10,000 to 20,000 rpm, and still moreillustratively approximately 15,000 to 18,000 rpm. For the Mabuchimotor, it has been found that 7.2V provides sufficient rpm for lysis. Itis understood, however, that the actual speed may be somewhat slowerwhen the blades 821 are impacting pouch 510. Other voltages and speedsmay be used for lysis depending on the motor and paddles used.Optionally, controlled small volumes of air may be provided into thebladder 822 adjacent lysis blister 522. It has been found that in someembodiments, partially filling the adjacent bladder with one or moresmall volumes of air aids in positioning and supporting lysis blisterduring the lysis process. Alternatively, other structure, illustrativelya rigid or compliant gasket or other retaining structure around lysisblister 522, can be used to restrain pouch 510 during lysis. It is alsounderstood that motor 819 is illustrative only, and other devices may beused for milling, shaking, or vortexing the sample.

Once the cells have been adequately lysed, the sample is moved throughchannel 538, blister 544, and channel 543, to blister 546, where thesample is mixed with a nucleic acid-binding substance, such assilica-coated magnetic beads 533. The mixture is allowed to incubate foran appropriate length of time, illustratively approximately 10 secondsto 10 minutes. A retractable magnet located within the instrumentadjacent blister 546 captures the magnetic beads 533 from the solution,forming a pellet against the interior surface of blister 546. The liquidis then moved out of blister 546 and back through blister 544 and intoblister 522, which is now used as a waste receptacle. One or more washbuffers from one or more of injection channels 515 c to 515 e areprovided via blister 544 and channel 543 to blister 546. Optionally, themagnet is retracted and the magnetic beads 533 are washed by moving thebeads back and forth from blisters 544 and 546 via channel 543. Once themagnetic beads 533 are washed, the magnetic beads 533 are recaptured inblister 546 by activation of the magnet, and the wash solution is thenmoved to blister 522. This process may be repeated as necessary to washthe lysis buffer and sample debris from the nucleic acid-bindingmagnetic beads 533, illustratively including 3 or more washes, althoughone wash may be sufficient for some embodiments disclosed herein and anynumber of washes is within the scope of this disclosure.

After washing, elution buffer stored at injection channel 515 f is movedto blister 548, and the magnet is retracted. The solution is cycledbetween blisters 546 and 548 via channel 552, breaking up the pellet ofmagnetic beads 533 in blister 546 and allowing the captured nucleicacids to dissociate from the beads and come into solution. The magnet isonce again activated, capturing the magnetic beads 533 in blister 546,and the eluted nucleic acid solution is moved into blister 548.

First-stage PCR master mix from injection channel 515g is mixed with thenucleic acid sample in blister 548. Optionally, the mixture is mixed byforcing the mixture between 548 and 564 via channel 553. After severalcycles of mixing, the solution is contained in blister 564, where apellet of first-stage PCR primers is provided, at least one set ofprimers for each target, and first-stage multiplex PCR is performed. IfRNA targets are present, an RT step may be performed prior to orsimultaneously with the first-stage multiplex PCR. First-stage multiplexPCR temperature cycling in the FilmArray® instrument is illustrativelyperformed for 15-20 cycles, although other levels of amplification maybe desirable, depending on the requirements of the specific application.The first-stage PCR master mix may be any of various master mixes, asare known in the art. In one illustrative example, the first-stage PCRmaster mix may be any of the chemistries disclosed in US2015/0118715,herein incorporated by reference, for use with PCR protocols taking 20seconds or less per cycle.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted, illustratively by forcing most of the sampleback into blister 548, leaving only a small amount in blister 564, andadding second-stage PCR master mix from injection channel 515 i.Alternatively, a dilution buffer from 515i may be moved to blister 566then mixed with the amplified sample in blister 564 by moving the fluidsback and forth between blisters 564 and 566. If desired, dilution may berepeated several times, using dilution buffer from injection channels515 j and 515 k, or injection channel 515 k may be reserved forsequencing or for other post-PCR analysis, and then adding second-stagePCR master mix from injection channel 515 h to some or all of thediluted amplified sample. It is understood that the level of dilutionmay be adjusted by altering the number of dilution steps or by alteringthe percentage of the sample discarded prior to mixing with the dilutionbuffer or second-stage PCR master mix comprising components foramplification, illustratively a polymerase, dNTPs, and a suitablebuffer, although other components may be suitable, particularly fornon-PCR amplification methods. If desired, this mixture of the sampleand second-stage PCR master mix may be pre-heated in blister 564 priorto movement to second-stage wells 582 for second-stage amplification.Such preheating may obviate the need for a hot-start component(antibody, chemical, or otherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 102 second-stage wells 582 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may bepre-loaded in the second-stage wells 582 as well. Each primer pair maybe similar to or identical to a first-stage PCR primer pair or may benested within the first-stage primer pair. Movement of the sample fromblister 564 to the second-stage wells 582 completes the PCR reactionmixture. Once high density array 581 is filled, the individualsecond-stage reactions are sealed in their respective second-stageblisters by any number of means, as is known in the art. Illustrativeways of filling and sealing the high density array 581 withoutcross-contamination are discussed in U.S. Pat. No. 8,895,295, alreadyincorporated by reference. Illustratively, the various reactions inwells 582 of high density array 581 are simultaneously thermal cycled,illustratively with one or more peltier devices, although other meansfor thermal cycling are known in the art.

In certain embodiments, second-stage PCR master mix contains the dsDNAbinding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate asignal indicative of amplification. However, it is understood that thisdye is illustrative only, and that other signals may be used, includingother dsDNA binding dyes and probes that are labeled fluorescently,radioactively, chemiluminescently, enzymatically, or the like, as areknown in the art. Alternatively, wells 582 of array 581 may be providedwithout a signal, with results reported through subsequent processing.

When pneumatic pressure is used to move materials within pouch 510, inone embodiment a “bladder” may be employed. The bladder assembly 810, aportion of which is shown in FIGS. 2-3, includes a bladder plate 824housing a plurality of inflatable bladders 822, 844, 846, 848, 864, and866, each of which may be individually inflatable, illustratively by acompressed gas source. Because the bladder assembly 810 may be subjectedto compressed gas and used multiple times, the bladder assembly 810 maybe made from tougher or thicker material than the pouch. Alternatively,bladders 822, 844, 846, 848, 864, and 866 may be formed from a series ofplates fastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 510 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

When pouch 510 is placed within the instrument 800, the bladder assembly810 is pressed against one face of the pouch 510, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 510. In addition to bladderscorresponding to many of the blisters of pouch 510, the bladder assembly810 may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch510. FIGS. 2-3 show an illustrative plurality of pistons or hard seals838, 843, 852, 853, and 865 that correspond to channels 538, 543, 553,and 565 of pouch 510, as well as seals 871, 872, 873, 874 that minimizebackflow into fitment 590. When activated, hard seals 838, 843, 852,853, and 865 form pinch valves to pinch off and close the correspondingchannels. To confine liquid within a particular blister of pouch 510,the hard seals are activated over the channels leading to and from theblister, such that the actuators function as pinch valves to pinch thechannels shut. Illustratively, to mix two volumes of liquid in differentblisters, the pinch valve actuator sealing the connecting channel isactivated, and the pneumatic bladders over the blisters are alternatelypressurized, forcing the liquid back and forth through the channelconnecting the blisters to mix the liquid therein. The pinch valveactuators may be of various shapes and sizes and may be configured topinch off more than one channel at a time. While pneumatic actuators arediscussed herein, it is understood that other ways of providing pressureto the pouch are contemplated, including various electromechanicalactuators such as linear stepper motors, motor-driven cams, rigidpaddles driven by pneumatic, hydraulic or electromagnetic forces,rollers, rocker-arms, and in some cases, cocked springs. In addition,there are a variety of methods of reversibly or irreversibly closingchannels in addition to applying pressure normal to the axis of thechannel. These include kinking the bag across the channel, heat-sealing,rolling an actuator, and a variety of physical valves sealed into thechannel such as butterfly valves and ball valves. Additionally, smallPeltier devices or other temperature regulators may be placed adjacentthe channels and set at a temperature sufficient to freeze the fluid,effectively forming a seal. Also, while the design of FIG. 1 is adaptedfor an automated instrument featuring actuator elements positioned overeach of the blisters and channels, it is also contemplated that theactuators could remain stationary, and the pouch 510 could betransitioned in one or two dimensions such that a small number ofactuators could be used for several of the processing stations includingsample disruption, nucleic-acid capture, first and second-stage PCR, andother applications of the pouch 510 such as immuno-assay and immuno-PCR.Rollers acting on channels and blisters could prove particularly usefulin a configuration in which the pouch 510 is translated betweenstations. Thus, while pneumatic actuators are used in the presentlydisclosed embodiments, when the term “pneumatic actuator” is usedherein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758 and 6,780,617, and U.S.Patent Application No. 2014/0038272, herein incorporated by reference.However, including the cell lysis within the sealed PCR vessel canimprove ease of use and safety, particularly if the sample to be testedmay contain a biohazard. In the embodiments illustrated herein, thewaste from cell lysis, as well as that from all other steps, remainswithin the sealed pouch. However, it is understood that the pouchcontents could be removed for further testing.

FIG. 2 shows an illustrative instrument 800 that could be used withpouch 510. Instrument 800 includes a support member 802 that could forma wall of a casing or be mounted within a casing. Instrument 800 mayalso include a second support member (not shown) that is optionallymovable with respect to support member 802, to allow insertion andwithdrawal of pouch 510. Illustratively, a lid may cover pouch 510 oncepouch 510 has been inserted into instrument 800. In another embodiment,both support members may be fixed, with pouch 510 held into place byother mechanical means or by pneumatic pressure.

In the illustrative example, heaters 886 and 888 are mounted on supportmember 802. However, it is understood that this arrangement isillustrative only and that other arrangements are possible. Bladderplate 810, with bladders 822, 844, 846, 848, 864, 866, hard seals 838,843, 852, 853, seals 871, 872, 873, 874 form bladder assembly 808 mayillustratively be mounted on a moveable support structure that may bemoved toward pouch 510, such that the pneumatic actuators are placed incontact with pouch 510. When pouch 510 is inserted into instrument 800and the movable support member is moved toward support member 802, thevarious blisters of pouch 510 are in a position adjacent to the variousbladders of bladder assembly 810 and the various seals of assembly 808,such that activation of the pneumatic actuators may force liquid fromone or more of the blisters of pouch 510 or may form pinch valves withone or more channels of pouch 510. The relationship between the blistersand channels of pouch 510 and the bladders and seals of assembly 808 isillustrated in more detail in FIG. 3.

Each pneumatic actuator is connected to compressed air source 895 viavalves 899. While only several hoses 878 are shown in FIG. 2, it isunderstood that each pneumatic fitting is connected via a hose 878 tothe compressed gas source 895. Compressed gas source 895 may be acompressor, or, alternatively, compressed gas source 895 may be acompressed gas cylinder, such as a carbon dioxide cylinder. Compressedgas cylinders are particularly useful if portability is desired. Othersources of compressed gas are within the scope of this invention.

Assembly 808 is illustratively mounted on a movable support member,although it is understood that other configurations are possible.

Several other components of instrument 810 are also connected tocompressed gas source 895. A magnet 850, which is mounted on a secondside 814 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 546 of pouch 510. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 546, and when magnet 850 is retracted, magnet 850 doesnot significantly affect any magnetic beads 533 present in blister 546.While reference is made to retracting magnet 850, it is understood thatan electromagnet may be used and the electromagnet may be activated andinactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field. Itis understood that the pneumatic connections may be pneumatic hoses orpneumatic air manifolds, thus reducing the number of hoses or valvesrequired.

The various pneumatic pistons 868 of pneumatic piston array 869 are alsoconnected to compressed gas source 895 via hoses 878. While only twohoses 878 are shown connecting pneumatic pistons 868 to compressed gassource 895, it is understood that each of the pneumatic pistons 868 areconnected to compressed gas source 895. Twelve pneumatic pistons 868 areshown.

A pair of heating/cooling devices, illustratively Peltier heaters, aremounted on a second side 814 of support 802. First-stage heater 886 ispositioned to heat and cool the contents of blister 564 for first-stagePCR. Second-stage heater 888 is positioned to heat and cool the contentsof second-stage blisters 582 of pouch 510, for second-stage PCR. It isunderstood, however, that these heaters could also be used for otherheating purposes, and that other heaters may be included, as appropriatefor the particular application.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 2, optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage well582 in pouch 510. Alternatively, camera 896 may take images that containall of the second-stage wells 582, and the image may be divided intoseparate fields corresponding to each of the second-stage wells 582.Depending on the configuration, optical array 890 may be stationary, oroptical array 890 may be placed on movers attached to one or more motorsand moved to obtain signals from each individual second-stage well 582.It is understood that other arrangements are possible.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Computer894 also controls heaters 886 and 888, and optical array 890. Each ofthese components is connected electrically, illustratively via cables891, although other physical or wireless connections are within thescope of this invention. It is understood that computer 894 may behoused within instrument 800 or may be external to instrument 800.Further, computer 894 may include built-in circuit boards that controlsome or all of the components, and may also include an externalcomputer, such as a desktop or laptop PC, to receive and display datafrom the optical array. An interface, illustratively a keyboardinterface, may be provided including keys for inputting information andvariables such as temperatures, cycle times, etc. Illustratively, adisplay 892 is also provided. Display 892 may be an LED, LCD, or othersuch display, for example.

EXAMPLES Example 1 High Density PCR

In one example, it is known that standard commercial immunofluorescenceassays for the common respiratory viruses can detect seven viruses:adenovirus, PIV1, PIV2, PIV3, RSV, Influenza A, and Influenza B. A morecomplete panel illustratively would include assays for other virusesincluding: coronavirus, human metapneumovirus, rhinovirus, and non-HRVenterovirus. For highly variable viruses such as Adenovirus or HRV, itis desirable to use multiple primers to target all of the branches ofthe virus' lineage (illustratively 4 outer and 4 inner primer setsrespectively). For other viruses such as coronavirus, there are 4distinct lineages (229E, NL63, OC43, HKU1) that do not vary from oneseason to another, but they have diverged sufficiently enough thatseparate primer sets are required. The FilmArray® Respiratory Panel(BioFire Diagnostics, LLC of Salt Lake City, Utah) includes Adenovirus,Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43,Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A,Influenza A/H1, Influenza A/H3, Influenza A/H1-2009, Influenza B,Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3,Parainfluenza Virus 4, and Respiratory Syncytial Virus. In addition tothese viruses, the FilmArray® Respiratory Panel includes three bacteria:Bordetella pertussis, Chlamydophila pneumonia, and Mycoplasma pneumonia.The high density array 581 is able to accommodate such a panel in asingle pouch 510. Other panels are available for the FilmArray®, eachassaying for at least 20 pathogens.

EXAMPLE 2: POUCH LOADING

FIG. 5 shows a loading station 600. As shown, pouch 510 of FIG. 1 hasbeen loaded into slot 610 of loading station 600, such that only fitment590 of pouch 510 is visible. As shown, loading station 600 is providedwith a sample vial receptacle 602 for holding sample vial 650 andhydration vial receptacle 604 for holding hydration vial 670. However,it is understood that the receptacles and vials are for aiding workflowand are illustrative only. Other configurations and use with otherpouches and other devices are within the scope of this disclosure.

A sample is pipetted or otherwise loaded into sample vial 650. Asdiscussed in more detail below, depending on work flow, sample vial 650may already contain a buffer or other fluid 652 for receiving thebiological sample, or the operator may add the biological sample in anappropriate buffer to sample vial 650. Optionally, the buffer may beprovided in a separate ampoule, with an appropriate amount of bufferapportioned. Similarly, hydration vial 670 may be preloaded with water,buffer, or other fluid 672, or the operator may load hydration vial 670with such fluid.

Illustrative fitment 590 includes an injection port 541 illustrativelyformed near second surface 595 of fitment 590. As shown, injection port541 is located in sample injection opening 563, which is configured toreceive a cannulated transfer vessel through first surface 594 offitment 590, such as a cannulated syringe. In this illustrativeconfiguration, injection port 541 is protected from accidental punctureand is not opened until a cannulated transfer vessel is placed intosample injection opening 563. Similarly, illustrative fitment 590includes a second injection port 588 illustratively formed near secondsurface 595 of fitment 590, and is located in hydration fluid injectionopening 583, which is configured similarly to sample injection opening563. As configured in this illustrative embodiment, injection port 541is for receiving the sample to be tested, which sample will be moved tochamber 592 a or directly into lysis blister 522 (FIG. 1), and secondinjection port 588 is configured for receiving the hydration fluid 672(displayed in FIG. 7), such as water or buffer, which hydration fluid672 will be moved to chambers 592 b through 592 l, for subsequentmovement through entry channels 515 b through 515 l. It is understoodthat the arrangement of injection ports 541 and 588 and openings 563 and583 is illustrative and that other configurations are within the scopeof this disclosure.

Illustrative sample vial 650, as best shown in FIG. 6, is comprised of atop surface 662, a vial body 654, and a cannula 655, in an arrangementsimilar to many cannulated syringes. In this illustrative embodiment,rather than the plunger found in many cannulated syringes, sample vial650 is provided with a cap 658 for extending through top surface 662 forsealing body 654. Illustratively, the operator would pour, pipette,insert swab, scoop solid or semi-solid material, or otherwise transfer afluid and/or other materials through opening 657 in top surface 662 andinto vial body 654.

Depending on the type of sample to be tested, sample vial 650 may beprovided with a filter 646, illustratively located at or near thehexagonal bottom surface 666 of vial body 654. As shown, filter 646 isheld in place by o-ring 644. However, it is understood that filter 646may be held in place by adhesive, by welding, by being press-fit intoplace, or by other means, as are known in the art. When cannula 655 isinserted into sample injection opening 563 and the sample is drawn intopouch 510, the sample material is filtered as it is pulled throughfilter 646 and into cannula 655. While the selection of filter materialdepends on the sample type and particle size, suitable filters forvarious biological samples include Pall 100 μm Absolute UltipleatPolypropylene Melt Blown Media and Millipore 80 μm Polypropylene NetFilter. Most syringe filters are designed to exclude organisms of acertain size, thereby removing those organisms from the filtrate. Unlikesuch pre-existing filters, these illustrative filters were chosen basedon their ability to exclude larger particulates found in stool, soil,powder, etc., while allowing target organisms (e.g., bacterial, viral,protozoan and fungal organisms) of approximately 60 μm in diameter orless to pass through in the filter. Also, the illustrative filtermaterial is inert (i.e. does not bind organism or nucleic acid) and isrelatively resistant to clogging. It is understood that theseillustrative filters were chosen for samples that include protozoans astarget organisms (up to about 60 μm). Because some pouch configurationsmay test only for smaller targets, filters with a smaller pore size maybe desired, such as filters with pore sizes of 1-10 μm for bacteria andfungi, and pore sizes of less than 1 μm if only viral particles are tobe detected. Of course, the larger pore size filter can still be used tofilter smaller targets. Such filters may be particularly useful forsample types that have a large amount of particulate matter, such assoil, stool, and powder that may clog the fluid system. Further, it isunderstood that the pore size is chosen based on the materials to befiltered, and that other pore sizes are within the scope of thisinvention.

It is understood that one or more components useful for samplepreparation may be provided dried in vial body 654. Such additives mayinclude buffering agents, stabilizers, proteases, DNAses, DNAseinhibitors, RNases, RNase inhibitors, lysozymes, reducing agents, andthe like. Alternatively, such components may be included in the samplebuffer, or may be added downstream, after the sample has exited vial 650for further processing. It is understood that the selection of suchadditives depends on the sample type and on the further processingdesired. Additives that help reduce viscosity or aid in solubility, toallow the sample to pass through filter 646 are particularly helpful.

As shown, bottom cap 664 is provided with a hexagonal portion 666, whichis configured to fit into the hexagonally shaped sample vial receptacle602. While hexagonal portion 666 and sample vial receptacle arehexagonal in the illustrative embodiment, it is understood that othershapes may be used, and that the hexagonal or other mating orinterlocking shapes may be provided to assist the operator in removingbottom cap 664. Alternatively, the operator may remove bottom cap 664 byother means, such as using two hands to twist bottom cap 664 from vialbody 654. Bottom cap 654 may be press-fit on, threaded onto, orotherwise affixed to vial body 654.

In the illustrative embodiment, bottom cap 664 is provided with a seat648, whereby a bottom end 659 of cannula 655 extends into seat 648.Illustratively, bottom end 659 of cannula 655 fits tightly into seat648, such that seat 648 provides an airtight seal around the open bottomend 659 of cannula 655. Optionally, vents 649 are provided betweenbottom cap 664 and vial body 654.

Turning now to FIG. 7, hydration vial 670 may be configured similarly tosample vial 650. However, it may be desirable to preload hydration vial670 with hydration fluid 672 and pre-seal the hydration fluid 672 inhydration vial 670, as shown in FIG. 7. Illustrative hydration vial 670,as shown in FIG. 7, is comprised of a top surface 682, a vial body 674,and a cannula 675, in an arrangement similar to that of sample vial 650.However, tongue 680 of cap 678 of illustrative hydration vial 670 isalready press-fit into opening 677 of top surface 682, and cap 678 maybe sealed to top surface 682, thereby preventing opening of hydrationvial 670. This arrangement is illustrative only, and it is understoodthat other ways of sealing hydration fluid 672 within hydration vial 670are envisioned herein. Illustratively, vial body 674 and cannula 675 maybe provided completely full or essentially completely full of fluid, sothat handling or rotating hydration vial 670 will not permit air toenter cannula 675. Alternatively, some air 685 or other gas may bepresent within vial body 674, and the operator may maintain hydrationbody in an upright position to prevent air from entering cannula 675. Inyet another alternative embodiment, the air 685 may be provided underpressure, and removal of bottom cap 684 would result in hydration fluidbeing forced through cannula 675. As shown, hydration vial 670 is notprovided with a filter, although one may be provided, if desired.

Bottom cap 684 may be provided to retain any fluid that might drip fromcannula 675, as well as preventing contamination of hydration fluid 672in cannula 675. A wiper 683 may be provided in bottom cap 684 to wipeexcess fluid from the bottom of cannula 675. The conical shape of wiper683 may also aid in retaining drips in bottom cap 684 during subsequenthandling and disposal. In the illustrative embodiment, bottom cap 684 isprovided with a hexagonal portion 686 for mating with the hexagonallyshaped hydration vial receptacle 604, although other shapes arepossible, as discussed above, with respect to sample vial 650. Hexagonalportion 686 of hydration vial 670 and hexagonally shaped hydration vialreceptacle 604 may be of different dimensions and/or different shapesthan hexagonal portion 666 of sample vial 650 and hexagonally shapedsample vial receptacle 602, such that only sample vial 650 will readilyfit into sample vial receptacle 602 and only hydration vial 670 willreadily fit into hydration vial receptacle 604, to reduce the chance ofthe operator confusing the sample vial 650 and hydration vial 670, sothat the proper fluids are injected through ports 541 and 588. Inaddition, sample vial 650 and injection opening 563 may be partially orentirely provided in a matching specific color, illustratively red,while hydration vial 670 and injection opening 583 may be partially orentirely provided in a different matching specific color, illustrativelyblue, to provide the operator with visual assistance in providing theproper fluids in ports 541 and 588. To further minimize risk ofinserting the wrong liquid into the wrong injection opening, thediameter of cannula 655 may differ from the diameter of cannula 675, andthe diameters of sample injection opening 563 and hydration fluidinjection opening 583 may similarly differ. Other configurations arewithin the scope of this disclosure.

Returning to FIG. 5, illustratively, to load pouch 510, the operatorwould place sample vial 650 into sample vial receptacle 602 andhydration vial 670 into hydration vial receptacle 604 on loading station600. Pouch 510 would also be placed into slot 610. The sample would beplaced into the sample buffer 652 in any way suitable for the sampletype, including inserting a swab 630, pipetting a fluid sample, drippingblood from a patient directly into the vial body, and placing a solid orsemi-solid sample such as stool into the vial body, with optionalvortexing or other mixing, as is standard in the art. Depending on thesample type and desired target nucleic acids, the sample buffer maycontain one or more additives or stabilizers, illustratively fortreating a biological or environmental sample, such as proteases,DNases, DNase inhibitors, RNases, RNase inhibitors, lysozymes, and thelike. Additionally or alternatively, these additives may be provided inthe pouch 510. Preferably before vortexing or mixing, the operator wouldclose sample vial 650 by placing the tongue 660 of cap 658 throughopening 657. Inserting tongue 660 pressurizes the air contained withinvial body 654. Illustratively, tongue 660 has a volume equal to orgreater than the volume of cannula 655. Illustratively, when bottom cap664 is removed, the airtight seal between seat 648 bottom end 659 ofcannula 655 is broken, and substantially all air is forced out ofcannula 655. If the volume of tongue 660 is greater than the volume ofcannula 655, such would help ensure that the maximal amount of air isdisplaced from cannula 655. Any overflow in the amount of fluid forcedinto and potentially through cannula 655 can be captured in bottom cap664 and removed from the bottom of cannula 655 by wiper 663. Bycompletely or essentially completely filling cannula 655, the quantityof bubbles in pouch 510 upon loading of the pouch is minimized. One ormore vents 649 may aid in separation of bottom cap 664 from hydrationvial 650.

Because bottom cap 664 is provided with a hexagonal portion 666, whichis configured to fit into the hexagonally shaped sample vial receptacle602, the operator can easily twist off bottom cap 654 while bottom capis engaging receptacle 602, thereby exposing cannula 655. Cannula 655 isthen inserted into sample injection opening 563 and is pushed in,opening injection port 541. A vacuum inside pouch 590 (or reducedpressure inside the pouch relative to atmospheric pressure or pressureoutside the pouch) illustratively forces the sample through the filter(if present), with or without pressure from the vial body, may be usedto draw the sample into pouch 510, illustratively into chamber 592 a infitment 590, for subsequent movement into lysis chamber 522. By assuringthat cannula 655 is substantially filled with fluid 652, the amount ofair or other gas moved from sample vial 650 into pouch 510 is minimized,thereby minimizing the size and quantity of bubbles. Furthermore, when aprior art syringe with a plunger is used and the vacuum inside pouch 590draws fluid, the plunger is drawn down the syringe, therebyequilibrating the pressure inside the syringe. In the embodiment ofFIGS. 5-6, because the opening at the top of each of the vial bodies issealed, when the vacuum from inside pouch 590 draws fluid from the vial,the vial will also experience negative pressure and may degas the sampleand draw some remaining air bubbles out of the pouch 590. Cannula 655 isthen withdrawn from sample injection opening 563 and sample vial 650 andbottom cap 664 are disposed of according to protocols. Since the vialbody 654 is under negative pressure, as cannula 655 is withdrawn, airbubbles that may have collected near injection port 541 may be drawn outof pouch 510, further reducing air bubbles in the pouch.

Similarly, the operator twists off bottom cap 684 from hydration vial670, thereby exposing cannula 675. If the contents of hydration vial 670are provided under pressure, a small amount of hydration fluid may leakout into bottom cap 684 when cannula 675 is separated from seat 692. Oneor more vents 693 may aid in separation of bottom cap 684 from hydrationvial 670. Cannula 675 is then inserted into hydration injection opening583 and is pushed in, opening injection port 588. Vacuum from insidefitment 590 may be used to draw the hydration fluid into pouch 510,illustratively into chambers 592 b-592 l, for subsequent movement intovarious blisters of pouch 510. Cannula 675 is removed from hydrationinjection opening 583, pouch 510 is removed from loading station 600 andplaced into instrument 800, and the run started. It is understood thatremoval of the vials is illustrative only. If the configuration of theinstrument and vials permit, the vials may be inserted permanently inthe injection ports, thereby becoming part of the closed system of thepouch and minimizing contamination from the sample. In such anembodiment, a seal bar may not be needed.

In the illustrative embodiment of sample vial 650 discussed above,tongue 660 has a volume equal to or greater than the volume of cannula655. In one exemplary embodiment where the pouch 510 has a fill volumeof 1 ml, vial body 654 may be provided with 1.5 ml of sample fluid 652and volume of 1 ml of air 645 above the sample fluid. Thus, the air is40% of the volume of the vial body 654. However, it is understood thatother percentages of air may be used, including 10%, 20%, 30% 50%, 60%,70%, 80%, and amounts in between. When tongue 660 is inserted throughopening 657, the air above the sample fluid is compressed,illustratively by about 50%, but compression in the range of 40-60%,30-70%, 20-80%, and 10-90% are all possible. It is understood thatchoice of volume of air and sample fluid depends on size of sample,diameter of cannula, whether removal of the vials prior to running thefluidic reaction is desired, and on a number of other factors. Forexample, scooped or swabbed samples may need a significantly largervolume of sample fluid, regardless of the fill volume of the fluidicsystem.

Illustrative vial bodies 654 and 674 are cylindrical. However, sincethese illustrative vials are provided without plungers, it is understoodthat the vial bodies need not have circular cross-sections, and that anybody shape is within the scope of this invention.

FIGS. 8-10 show an alternative embodiment to loading station 600 andvials 650, 670, with like numbers indicating similar parts. Loadingstation 700, as shown in FIG. 8, may be similar to loading station 600,with sample vial receptacle 702 and hydration vial receptacle 704, andslot 710 for receiving pouch 510, similar to those shown in FIG. 1.However, according to at least one embodiment, the shape and location ofthe receptacles are significantly different between loading station 600and loading station 700. For instance, in at least one embodiment, ascompared to receptacles 602, 604 of loading station 600, receptacles 702and 704 are closer to pouch 510. With this reduced distance, there isless opportunity for drips to occur upon loading pouch 510. Furthermore,as best seen in FIGS. 9-10, bottom cap 764 of sample vial 750 isprovided with four relatively short fins 767 that fit within fourmatching slots 703 of sample vial receptacle 702, and bottom cap 784 ofhydration vial 770 is provided with two relatively longer fins 787 thatfit within two matching slots 705 of hydration vial receptacle 704.These fins replace the hexagonal portions 666 and 686 of vials 650 and670, respectively. The larger number of fins 767 on bottom cap 764prevents sample vial 750 from being placed in hydration vial receptacle704, and the longer fins 787 of bottom cap 784 prevents hydration vial770 from being placed in sample vial receptacle 702. However, it isunderstood that the use of fins of different sizes and numbers isillustrative only, and that different keying systems are within thescope of this disclosure. As discussed above with respect to loadingstation 600, the receptacles 702, 704 of loading station 700 may be usedto assist with twisting off bottom caps 764, 784 from their respectivevial bodies 754, 774, to aid with the loading process.

While sample vials 650, 750 and hydration vials 670, 770 are used in theillustrative example for loading of pouch 510, it is understood thatthese loading vials are suitable for loading any of the pouchesdisclosed herein. They are also suitable for loading other fluidic ormicrofluidic device, especially fluidic devices that are configured todraw liquid into the fluidic device using vacuum or suction.

Example 3 Sample Preparation

In this illustrative embodiment, samples from lower respiratory tractinfections (LRTI) are used. However, it is understood that the presentmethods may be suitable for other sample types, including but notlimited to difficult-to-process biological and environmental sampletypes, and is particularly useful for any sample type that isselectively bound and then released from the collection material. LRTIsare common ailments, but identification of the etiological agent can bedifficult as a result of biased diagnostic techniques and inhibitoryspecimen characteristics. The current “Gold Standard” for pathogenidentification is bacterial culture, which is highly subjective, lackssensitivity, and is an incomplete screening method, as culture is onlycapable of identifying culturable pathogens, whereas non-culturablepathogens go undetected. LRTI specimens include multiple specimen typessuch as broncho-alveolar lavage (BAL), mini-BAL, bronchial washings,sputum, and endotracheal aspirates (ETAs), all of which have unique andoften challenging characteristics. Sputum and ETA specimens can bedifficult to manipulate, can be slow to process, and may be highlyinhibitory toward molecular detection methods. Additionally, LRTIpathogens include gram-positive and gram-negative bacteria, viruses, andfungal organisms, often requiring a variety of sample processingmethods.

Sputum is a semi-solid sample matrix, with variable viscosity andparticulates. BALs can have a viscosity similar to clean saline or canhave a viscosity similar to sputum, both of which can present processingchallenges. Difficulties include un-pipetable specimens, inaccuratepipetting volumes due to variable viscosity, air pockets, pull-back,remnants, and foaming during pouch injection or sample manipulation.With the high viscosity, it can be difficult to isolate pathogens fromthe sample matrix. In addition, RNase activity found in such samples canaffect RNA detection. With such sample types, a combination of physicaland chemical lysis can be effective.

The sputum sample may be obtained using any suitable means, includinghaving the patient expectorate into a suitable collection vessel orinducing the patient to produce a specimen. Most pathogen detectionmethods use a volume transfer system that can be difficult to use withsputum or other viscous samples. Typically, the sample is pre-treated toliquidize the sample for subsequent pipetting. In one illustrativeembodiment, swabs are used to transfer sample, reducing or eliminatedthe need for a pre-treatment step. While pipets, other swabs, and othertransfer devices may be used for transfer of the sample, use of flockedswab provides a simple and easy method to introduce a specimen into thetesting system while minimizing issues with specimen manipulation. Onesuitable swab is FLOQSwabs™ (Copan Diagnostics, Murrieta, Calif.). Thisillustrative flocked swab is hydrophilic and is well suited to collectpathogens. For such viscous samples, illustratively having a viscosityof 3 cP or greater, or solid or semi-solid samples, the flocked swab maypreferentially collect organism over solid material. The flocked swabmay also preferentially release organism over sample matrix material.Other materials that preferentially collect and release organism oversample matrix may be used to transfer the sample and it is understoodthat a swab format is illustrative only.

In one illustrative embodiment, the swab 630 may be placed into thesample collected from the patient or may be placed into an environmentalsample. The swab 630 then may be placed directly into sample buffer 652in sample vial 650. Sample buffer 652 may be any buffer, depending onthe sample type. As shown in FIG. 6, if desired, the tip of the swab 630may be broken off and cap 658 may be used to close vial body 654. Thepresence of the swab tip 630 within vial body 654 should notsignificantly affect the use of vial 650. Vial 650 may be shaken,illustratively by inverting three times, or shaken more vigorouslyillustratively for 10 seconds, to release any pathogens that are presentfrom the swab 630. Alternatively, swab 630 may be swirled in the samplebuffer 652 and then swab 630 may be removed from vial 650. While notbeing bound to theory, it is believed that flocked swabs canpreferentially collect cells, organisms, and viruses over samplematrices, and then may retain a portion of the sample matrices such asviscous sputum material while at least releasing organisms and viruses.However, it is understood that other swabs or transfer devices may beused in various embodiments. Illustratively, any transfer material thatselectively collects and releases the desired sample over the samplematrix can be used to concentrate the sample. Such materials includeswabs, brushes, sponges, filters, utensils, loops, paper, or otheradsorbent material that selectively binds and releases the desiredsample and can be placed in contact with the sample during transfer ofthe sample for analysis.

Thus, it has been found that such a flocked swab is suitable for bothcollection and release of pathogens in viscous samples such as LRTIsamples, and the flocked swab provides a fairly uniform collectionvolume. A single FLOQSwabs™ swab collects approximately 150 to 300 μl ofsample, including higher viscosity sample types such as sputum, in afairly reproducible manner. Other materials that reproducibly collectviscous samples with a small sample volume range, illustratively atwo-fold to four-fold volume range, are within the scope of theinvention. It is understood that a sample volume range is the range ofvolumes that a sample collection material reproducibly collects, suchthat an illustrative two-fold range is 150 to 300 μl and an illustrativefour-fold range is 150 to 600 μl, although other ranges, including othertwo-fold and four-fold ranges are within the scope of the invention.Illustrative examples include swabs that are sized to collect 50 to 150μl, as well as 300 to 1000 μl of sample, and possibly 5 to 10 ml,although it is understood that these ranges are illustrative and thatother size ranges are possible. While such swabs may be used to collectspecimens from a patient, in one illustrative embodiment, the swab isused collect the specimen in a reproducible manner and to transfer thesample from a suitable collection vessel to another vessel for furtherprocessing. However, it is understood that other suitable collection andtransfer materials may be used with various embodiments disclosedherein, as are known in the art.

It is known in the art to pretreat difficult samples for a period oftime. For example, sputum may be treated with dithiothreitol (DTT)and/or heat for 15 minutes or more, sometimes 30 minutes or more. Asample buffer 652 may be used that may partially or completely liquefysputum-like specimens, illustratively without such a pretreatment.Buffers known in the art often use detergents at amounts of 10% or lessby volume. An illustrative sample buffer 652 according to the presentinvention may contain 10% or more detergent, illustratively 12%, 15%,18%, 20% or more detergent. Suitable detergents include non-ionic, ionicand zwitterionic detergents may be used. In one illustrative embodiment,a non-ionic detergent such as 15% Triton-X may be used. Othernon-limiting detergents include Tween-20, SDS, and NP-40. It has beenfound that this higher concentration of detergent may reduce oreliminate the need for a pretreatment such as heating the sample or aDTT mixture pretreatment, prior to nucleic acid extraction. Thus, thesample may be processed with only 5 minutes or less, 2 minutes or less,one minute or less, or even without a hold time. However, it isunderstood that a pretreatment may be desirable with some sample types.The sample buffer 652 may also contain a guanidinium compound, such asguanidine hydrochloride. In one illustrative example, the buffer isacidic, illustratively with a pH from about 2 to about 6. In certainsample preparation methods, illustratively when the sample type is bloodor a blood component, an alcohol such as isopropanol is used in thesample buffer 652. However, other sample buffers may be alcohol-free. Ithas been found that in various illustrative embodiments employingdetergents at a concentration of 10% or more, illustratively 15%, thealcohol may be omitted from the sample buffer 652.

As discussed above, sample vial 650 may be provided with filter 646.When cannula 655 is inserted into sample injection opening 653 and thesample is drawn into pouch 510, the sample material is filtered as it ispulled through filter 646 and into cannula 655. Such a filter 646 mayclog with viscous samples such as sputum. In some embodiments, samplevial 650 may be provided with a protease, such as Proteinase K, or theprotease may be otherwise provided in sample vial 650 or may be addedalong with sample buffer 652. If the protease is provided in dried form,introduction of sample buffer 652 rehydrates the protease, andintroduction of the sample into sample buffer 652 allows the protease tobegin to break down the proteins in the sample, even before the sampleis transferred into pouch 510 and reaches lysis blister 522. Action ofthe protease may aid in reducing the viscosity of the sample, therebyimproving the sample's ability to pass through filter 646, or improveother downstream processing steps. Thus, more of the pathogens may beliberated from the sample with protease present in the sample vial 650.However, in some embodiments using a protease, it may be desirable toadd the protease into the sample buffer directly or add the proteaselater in the process, illustratively by having it dried in entry channel515 b, while in other embodiments, the protease may be omitted.

It is understood that preferentially transferring the pathogens by usinga flocked swab, higher detergent concentration, filtering through afilter 646 in the presence of a protease, or a combination thereof mayobviate the need for a heat or DTT pre-treatment and may break up suchviscous samples sufficiently. Such samples may then be ready for asubsequent lysis step, followed by PCR or other testing.

While the above illustrative description focuses on sputum and otherLRTI sample types, it is understood that the above methods and devicesmay be used with a wide variety of sample types, particularly, but notlimited to, difficult sample types. In addition to sputum, BAL, andother LRTI sample types, other difficult biological samples include butare not limited to mucus, stool, tissue, tissue homogenate, groundtissue, paraffin treated formalin embedded tissue, bone, bonehomogenate, eschars, puss, synovial fluid, lymph node aspirates, andstomach washings. Environmental samples, illustratively soil, surfaces,powders or food, may also present challenges. Transferring using aflocked swab or other transfer means that preferentially collect andrelease pathogens allows for relatively consistent collection of thepathogens, even from samples that are difficult to pipet or measureaccurately. A sample buffer that partially or completely liquefies thesample matrix may be used. Drawing the sample through a filter,illustratively in the presence of higher concentrations of detergent oradditives to aid in sample preparation, helps to break up the samplematrix to provide a sample in a format suitable for further processing.

Example 4 Direct Blood Samples

In this embodiment direct blood samples are used for host RNA analysis.As used herein, “direct blood” means blood samples that have not beencultured. Illustrative direct blood samples include whole blood directfrom a host organism, illustratively a human, a whole blood fraction(e.g. plasma, or serum), whole blood collected in an anticoagulantsolution (e.g. EDTA, Citrate), or whole blood collected in a RNAstabilizing solution. It is understood that host response to infectious,acute, and/or chronic diseases results in changes in production ofcertain RNA molecules, including but not limited to ribosomal RNA,messenger RNA (mRNA), long noncoding RNA, or small interfering RNA. Suchchanges in RNA production may be used to indicate the source of thehost's illness. The RNA in a direct blood sample is generally purifiedwith automated or manual extraction methods comprised of centrifugation,washing, addition of optimized buffers, protein and nucleic aciddigests, often with additional washing, and elution. In many prior artmethods, this is all completed using equipment separate from the endpoint molecular diagnostic tool and often requires 90 minutes or more.The resultant purified samples are then manually added to an end pointmolecular diagnostic system such as a cDNA microarray or a qPCRinstrument. The process for purifying host RNA samples often requiressignificant time, external equipment, and sample manipulation, all ofwhich can influence sample integrity and quality. An alternativeapproach is to add the direct blood sample (illustratively in any of theforms defined above) directly to a sample buffer 652. An illustrativesample buffer contains a chaotropic agent that assists in the bindingand recovery of nucleic acids, while reducing proteins like RNAses thatcan degrade RNA. Illustratively, the ratio of direct blood sample tosample buffer is in a range from 1 to 8 to 1 to 1, more specifically ina range from 1 to 5 to 1 to 1.67, although it is understood that otherratios are possible. This sample may then be loaded directly into pouch510, as discussed above. Host RNA is extracted and purified within theclosed system, as discussed above, illustratively with only minimalmixing in vial 650 or without any additional user manipulation. Suchsample prep can be completed without any or all of steps involvingcentrifugation, ethanol precipitation, and DNA digestion. Thus, thesample may be processed with only 2 minutes or less and require noexternal equipment from the end point molecular diagnostic tool.

An example of this application is the detection of host RNA changes inresponse to an influenza infection. One-hundred microliters of (100 μL)whole blood sample collected in a RNA stabilizing solution according tomanufacturer instructions was added to sample buffer 652, illustrativelythe sample buffer provided with the FilmArray Respiratory Panel (BioFireDiagnostics, LLC). Illustratively, 300 to 900 μl, or 2-5 fold the volumeof the sample of sample buffer may be used. This mixture was theninjected into a disposable pouch 510. Sample extraction, purification,reverse-transcription, and two stage of polymerase chain reactionresulted in measurements of significantly upregulated host RNA inresponse to viral infection relative to a healthy control. The entireprocess of sample purification and qPCR analysis took less than 70minutes.

A further example of this application is the detection of host RNA fromwhole blood collected in an anticoagulant solution (EDTA). One-hundredmicroliters of (100 μL) this whole blood sample was added to 800 μL ofthe same sample buffer. This mixture was then injected into a pouch 510.Sample extraction, purification, reverse-transcription, and two stagesof polymerase chain reaction resulted in measurements host RNA. Theentire process of sample purification and qPCR analysis took less than70 minutes.

These examples demonstrate that nucleic acids, illustratively high copynumber nucleic acids including but not limited to various RNA molecules,bacteria, viruses, or other microorganisms, may be extracted from directblood samples using only lysis, illustratively chemical and/ormechanical lysis, and nucleic acid purification, illustratively bybinding to and eluting from a silica substrate, illustratively no morethan 5 washes, or no more than 3 washes, but as few as one wash iswithin the scope of this invention. These nucleic acids may be extractedand prepared without centrifugation or enzymatic digestion, and withoutmany repeated washing steps. Also as demonstrated, these extractednucleic acids are suitable as template for subsequent amplification.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Whilecertain embodiments and details have been included herein and in theattached invention disclosure for purposes of illustrating theinvention, it will be apparent to those skilled in the art that variouschanges in the methods and apparatus disclosed herein may be madewithout departing from the scope of the invention, which is defined inthe appended claims. All changes which come within the meaning and rangeof equivalency of the claims are to be embraced within their scope.

1. A method of amplifying RNA from a direct blood sample comprising: (a)adding the direct blood sample to a sample buffer, (b) bead beating thesample buffer, (c) extracting the RNA from the sample buffer, and (d)amplifying the RNA.
 2. The method of claim 1, wherein steps (b) through(d) are performed in a closed sample vessel.
 3. The method of claim 1,wherein steps (a) through (d) are performed without centrifugation,ethanol precipitation, and DNA digestion.
 4. The method of claim 1,wherein the RNA is host RNA.
 5. The method of claim 1, wherein thedirect blood sample is collected in a RNA stabilized solution.
 6. Themethod of claim 1, wherein the direct blood sample is collected in anon-RNA stabilizing solution.
 7. The method of claim 1, wherein thedirect blood sample is separated prior to step (a) and a blood fractionis used.
 8. The method of claim 1, further comprising filtering thesample between steps (a) and (b).
 9. The method of claim 1, wherein theRNA is provided in high copy number.
 10. The method of claim 1, whereinthe RNA is from microorganisms.
 11. The method of claim 1, furthercomprising washing the RNA following the extracting step, wherein thewashing step comprises no more than three washes.
 12. The method ofclaim 1, wherein the sample buffer includes a guanidinium compound. 13.The method of claim 1, wherein step (g) includes a first-stage multiplexPCR and a plurality of individual second-stage PCR reactions.